WO2021247794A2

WO2021247794A2 - B7h3-targeting proteins and methods of use thereof

Info

Publication number: WO2021247794A2
Application number: PCT/US2021/035591
Authority: WO
Inventors: Martin FELICES; Jeffrey S. Miller
Original assignee: Regents Of The University Of Minnesota
Priority date: 2020-06-03
Filing date: 2021-06-03
Publication date: 2021-12-09
Also published as: WO2021247794A3; JP2023529368A; BR112022024768A2; CA3183711A1; KR20230019949A; CN115968378A; AU2021283355A1; US20230203166A1; IL298641A; EP4161967A2

Abstract

Multispecific B7H3 targeting compounds and methods of use thereof are provided. The multispecific compounds include bispecific targeting proteins including a B7H3 targeting domain and an immune cell engaging domain; and trispecific targeting proteins including a B7H3 targeting domain, an immune cell engaging domain and an immune cell activating domain. The methods of use include methods of inducing NK-mediates cell killing, methods of inducing NK expansion in vivo, and methods of treating cancer.

Description

B7H3-TARGETING PROTEINS AND METHODS OF USE THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority under 35 U.S.C. §119(e) of U.S. Provisional Application No. 63/033,989, filed June 3, 2020, which is incorporated by reference in its entirety.

SEQUENCE LISTING

This application contains a Sequence Listing electronically submitted via EFS-Web to the United States Patent and Trademark Office as an ASCII text file entitled “0110- 000661WO01_ST25.txt” having a size of 40 kilobytes and created on June 2, 2021. The information contained in the Sequence Listing is incorporated by reference herein.

SUMMARY

In one aspect, this disclosure describes an anti-B7H3 protein that includes at least one of SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6, or a functional variant thereof.

In another aspect, this disclosure describes an anti-B7H3 protein that includes SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3 a CDR region of SEQ ID NO: 1, a CDR region of SEQ ID NO:2, a CDR region of SEQ ID NO:3, or a functional variant thereof.

In another aspect, this disclosure describes a multispecific compound. Generally, the multispecific compound includes a targeting domain and an immune cell engaging domain operably linked to the targeting domain. The targeting domain includes an anti-B7H3 protein.

In some embodiments, the immune cell is a T cell or a Natural Killer (NK) cell. In embodiments in which the immune cell is an NK cell, the immune cell engaging domain can include a ligand or antibody that specifically binds to CD 16. In embodiments in which the immune cell engaging domain includes an antibody that specifically binds to CD 16, the antibody can be an scFv, a F(ab)₂, a Fab, or a single domain antibody (sdAb).

In some embodiments, the immune cell engaging domain can include SEQ ID NO: 19. In some embodiments, the targeting domain can include SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3. In some embodiments, the immune cell engaging domain can include SEQ ID NO: 19 and the targeting domain can include SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3.

In some embodiments, the immune cell engaging domain and the targeting domain are linked by a linker having the amino acid sequence of any one of SEQ ID NOs: 12-18. In some of these embodiments, the immune cell engaging domain and the targeting domain are linked by a linker having the amino acid sequence of SEQ ID NO: 14.

In some embodiments, the anti-B7H3 multispecific compound includes amino acids 19- 294 of SEQ ID NO:20 or amino acids 19-284 of SEQ ID NO:21.

In some embodiments, the anti-B7H3 multispecific compound can be a trispecific compound that further include an immune cell activating domain. In embodiments in which the immune cell is an NK cell, the immune cell activating domain includes a cytokine or a functional portion thereof. In some of these embodiments, the cytokine is IL-15 or a functional variant thereof.

In some embodiments, the trispecific compound can include the amino acids of SEQ ID NO: 19 as the immune cell engaging domain, the amino acids of SEQ ID NO: 19 as the immune cell activating domain, and the amino acids of any one of SEQ ID NOs: 1-3 as the targeting domain. The functional domains may be linked by any one or combination of two or more linkers reflected in SEQ ID NOs: 12-18. In some of these embodiments, the immune cell engaging domain may be linked to the immune cell activating domain by a linker having the amino acid sequence of SEQ ID NO: 14. In some embodiments, the immune cell activating domain and the targeting domain may be linked by a linker having the amino acid sequence of SEQ ID NO: 15.

In some embodiments, the anti-B7H3 trispecific compound can include the amino acid sequence of SEQ ID NO:22 or SEQ ID NO:23.

In some embodiments, the functional variant of IL-15 includes an N72D or N72A amino acid substitution compared to SEQ ID NO: 11.

In another aspect, this disclosure describes an isolated nucleic acid sequence encoding any embodiment of the anti-B7H3 multispecific compound described herein.

In another aspect, this disclosure describes a host cell that includes any embodiment of the isolated nucleic acid summarized above. In some embodiments, the host cell is a T cell, an NK cell, or a macrophage. In another aspect, this disclosure describes a pharmaceutical composition that includes an anti-B7H3 multispecific compound and a pharmaceutically acceptable carrier.

In another aspect, this disclosure describes a method that generally includes administering to a subject an anti-B7H3 multispecific compound in an amount effective to induce natural killer (NK)-mediated killing of a cell. The anti-B7H3 multispecific compound includes a targeting domain that includes the anti-B7H3 protein, and an NK engaging domain operably linked to the targeting domain.

In another aspect, this disclosure describes a method for stimulating expansion of natural killer (NK) cells in vivo. Generally, the method includes administering to a subject an effective amount of an anti-B7H3 multispecific compound to the subject. The anti-B7H3 multispecific compound includes a targeting domain that includes the anti-B7H3 protein, and an NK engaging domain operably linked to the targeting domain.

In another aspect, this disclosure describes a method of treating a subject having, or at risk of having cancer. Generally, the method includes administering to a subject an effective amount of an anti-B7H3 multispecific compound to the subject. The anti-B7H3 multispecific compound includes a targeting domain that includes the anti-B7H3 polypeptide, and an NK engaging domain operably linked to the targeting domain. In some embodiments, the cancer cells express B7H3.

In another aspect, this disclosure describes a chimeric antigen receptor compound that includes an anti-B7H3 polypeptide.

In another aspect, this disclosure describes a targeted therapeutic compound that includes a targeting domain and a therapeutic domain linked to the targeting domain. The targeting domain includes an anti-B7H3 polypeptide. In some embodiments, the targeted therapeutic provides targeted immunotherapy. In some embodiments, the therapeutic domain includes a drug, a therapeutic radioisotope, a toxin, a cytokine, or a chemokine.

In another aspect, this disclosure describes a targeted imaging compound that includes a targeting domain and an imaging domain linked to the targeting domain. The targeting domain includes an anti-B7H3 polypeptide. In some embodiments, the imagining domain includes a colorimetric label, a fluorescent label, a radioactive label, a magnetic label, or an enzymatic label. In another aspect, this disclosure describes a capture assay device that includes an anti- B7H3 polypeptide immobilized to a substrate.

BRIEF DESCRIPTION OF THE DRAWINGS The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. FIGURE 1A -1B illustrate schematic of trispecific killer engager incorporating exemplary embodiments of anti-B7H3 protein. FIGURE 1A illustrates a schematic of an exemplary anti-B7H3 protein sequence (e.g., a sdAb) incorporated into the trispecific killer engager backbone, which also includes recombinant human IL-15 and an anti-CD 16 single domain antibody (sdAb) arm, all connected through short linkers. FIGURE IB illustrates the amino acid sequences for the novel anti-B7H3 proteins.

FIGURES 2A-2F illustrate NK cell degranulation and interferon gamma (IFN-γ) production measured by flow cytometry. FIGURE 2A is a graph bar illustrating NK cell degranulation (%CD107a+) when trispecific killer engagers (30 nM) were incubated with peripheral blood mononuclear cells (PBMCs) alone. FIGURE 2B is a graph bar illustrating NK cell degranulation when trispecific killer engagers (30 nM) were co-cultured with PBMCs and prostate cancer cells (PC3). FIGURE 2C is a graph bar illustrating NK cell degranulation when trispecific killer engagers (30 nM) were co-cultured with PBMCs and prostate cancer cells (DU145). FIGURE 2D is a graph bar illustrating IFN-γ production when trispecific killer engagers (30 nM) were incubated with PBMCs alone. FIGURE 2E is a graph bar illustrating IFN-γ production when trispecific killer engagers (30 nM) were co-cultured with PBMCs and PC3 cells. FIGURE 2F is a graph bar illustrating IFN-γ production when trispecific killer engagers (30 nM) were co-cultured with PBMCs and DU145 cells.

FIGURES 3A-3D illustrate NK cell degranulation and IFN-γ production measured by flow cytometry. FIGURE 3 A is a graph bar illustrating NK cell degranulation (%CD107a+) when trispecific killer engagers (30 nM) were co-cultured with PBMCs and prostate cancer cells (LnCAP). FIGURE 3B is a graph bar illustrating NK cell degranulation when trispecific killer engagers (30 nM) were co-cultured with PBMCs and prostate cancer cells (C4-2). FIGURE 3C is a graph bar illustrating IFN-γ production when trispecific killer engagers (30 nM) were co- cultured with PBMCs and LnCAP cells. FIGURE 3D is a graph bar illustrating IFN-γ production when trispecific killer engagers (30 nM) were co-cultured with PBMCs and C4-2 cells.

FIGURES 4A-4F illustrate NK cell degranulation and IFN-γ production measured by flow cytometry. FIGURE 4A is a graph bar illustrating NK cell degranulation when trispecific killer engagers (30 nM) were incubated with PBMCs alone. FIGURE 4B is a graph bar illustrating NK cell degranulation when trispecific killer engagers (30 nM) were co-cultured with PBMCs and C4-2 cells. FIGURE 4C is a graph bar illustrating NK cell degranulation when trispecific killer engagers (30 nM) were co-cultured with PBMCs and lung cancer cells (A549). FIGURE 4D is a graph bar illustrating IFN-γ production when trispecific killer engagers (30 nM) were incubated with PBMCs alone. FIGURE 4E is a graph bar illustrating IFN-γ production when trispecific killer engagers (30 nM) were co-cultured with PBMCs and C4-2 cells. FIGURE 4F is a graph bar illustrating IFN-γ production when trispecific killer engagers (30 nM) were co- cultured with PBMCs and A549 cells.

FIGURES 5 A-5F illustrate NK cell degranulation and IFN-γ production measured by flow cytometry. FIGURE 5A is a graph bar illustrating NK cell degranulation when trispecific killer engagers (30 nM) were co-cultured with PBMCs and lung cancer cells (NCI-H460s). FIGURE 5B is a graph bar illustrating NK cell degranulation when trispecific killer engagers (30 nM) were co-cultured with PBMCs and ovarian cancer cells (OVCAR8). FIGURE 5C is a graph bar illustrating NK cell degranulation when trispecific killer engagers (30 nM) were co-cultured with PBMCs and ovarian cancer cells (MAI48). FIGURE 5D is a graph bar illustrating IFN-γ production when trispecific killer engagers (30 nM) were co-cultured with PBMCs and NCI- H460s cells. FIGURE 5E is a graph bar illustrating IFN-γ production when trispecific killer engagers (30 nM) were co-cultured with PBMCs and OVCAR8 cells. FIGURE 5F is a graph bar illustrating IFN-γ production when trispecific killer engagers (30 nM) were co-cultured with PBMCs and MAI48 cells.

FIGURES 6A-6F illustrate NK cell degranulation measured by flow cytometry. FIGURE 6A is a graph bar illustrating NK cell degranulation when trispecific killer engagers (0.3 nM) were incubated with PBMCs alone. FIGURE 6B is a graph bar illustrating NK cell degranulation when trispecific killer engagers (3 nM) were incubated with PBMCs alone. FIGURE 6C is a graph bar illustrating NK cell degranulation when trispecific killer engagers (30 nM) were incubated with PBMCs alone. FIGURE 6D is a graph bar illustrating NK cell degranulation when trispecific killer engagers (0.3 nM) were co-cultured with PBMCs and C4-2 cells. FIGURE 6E is a graph bar illustrating NK cell degranulation when trispecific killer engagers (3 nM) were co-cultured with PBMCs and C4-2 cells. FIGURE 6F is a graph bar illustrating NK cell degranulation when trispecific killer engagers (30 nM) were co-cultured with PBMCs and C4-2 cells.

FIGURES 7A-7F illustrate IFN-γ production measured by flow cytometry. FIGURE 7A is a graph bar illustrating IFN-γ production when trispecific killer engagers (0.3 nM) were incubated with PBMCs alone. FIGURE 7B is a graph bar illustrating IFN-γ production when trispecific killer engagers (3 nM) were incubated with PBMCs alone. FIGURE 7C is a graph bar illustrating IFN-γ production when trispecific killer engagers (30 nM) were incubated with PBMCs alone. FIGURE 7D is a graph bar illustrating IFN-γ production when trispecific killer engagers (0.3 nM) were co-cultured with PBMCs and C4-2 cells. FIGURE 7E is a graph bar illustrating IFN-γ production when trispecific killer engagers (3 nM) were co-cultured with PBMCs and C4-2 cells. FIGURE 7F is a graph bar illustrating IFN-γ production when trispecific killer engagers (30 nM) were co-cultured with PBMCs and C4-2 cells.

FIGURE 8 shows photographs illustrating the enhancement by trispecific killer engager incorporating exemplary anti-B7H3 protein of cytolytic activity against ovarian cancer spheroids.

FIGURE 9 is a graph illustrating the quantification of the enhancement by trispecific killer engager incorporating exemplary anti-B7H3 protein of cytolytic activity against ovarian cancer spheroids.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

This disclosure describes anti-B7H3 polypeptides, compounds and devices that include anti-B7H3 polypeptides, and methods of suing such compounds and devices. Exemplary platforms in which an anti-B7H3 polypeptide may be used include, but are not limited to, chimeric antigen receptor therapies (e.g., CAR-NK therapy, CAR-T therapy, CAR-macrophage therapy, etc.), multispecific immune cell engager technologies (e.g., bispecific killer engagers, trispecific killer engagers, bispecific T cell engagers, trispecific T cell engagers, etc.), targeted immunotherapies (e.g., targeted ADAM17 blocker (TAB) therapy), delivery of therapeutics (e.g., antibody-drug conjugates, delivery of therapeutic radioisotopes, delivery of toxins, delivery of cytokines, delivery of chemokines), imaging technologies (delivery of labeling constructs and/or labeling radioisotopes), cell and/or ligand capture technologies (e.g., ELISA, etc.)

B7 Homolog 3 (B7H3), also known as cluster of differentiation 276 (CD276), is a human protein encoded by the CD276 gene. The B7H3 protein is a 316 amino acid-long type I transmembrane protein existing in two isoforms determined by its extracellular domain. In mice, the extracellular domain consists of a single pair of immunoglobulin variable (IgV)-like and immunoglobulin constant (IgC)-like domains, whereas in humans it consists of one pair (2Ig- B7H3) or two identical pairs (4Ig-B7H3) due to exon duplication. B7H3 mRNA is expressed in most normal tissues. In contrast, B7H3 protein has a very limited expression on normal tissues because of its post-transcriptional regulation by microRNAs. In normal tissues, B7H3 has a predominantly inhibitory role in adaptive immunity, suppressing T cell activation and proliferation.

B7H3 is overexpressed among several kinds of human cancer cells. In malignant tissues, B7H3 is an immune checkpoint molecule that inhibits tumor antigen-specific immune responses. B7H3 also possesses non-immunological pro-tumorigenic functions such as promoting migration, invasion, angiogenesis, chemoresistance, epithelial-to-mesenchymal transition, and affecting tumor cell metabolism. B7H3 is recognized as a co-stimulatory molecule for immune reactions including, but not limited to, T cell activation and IFN-γ production. In the presence of anti-CD3 antibody mimicking the TCR signal, human B7H3-Ig fusion protein increases the proliferation of both CD4⁺ and CD8⁺ T cells and enhances the cytotoxic T lymphocyte (CTL) activity in vitro. B7H3 also has an antitumor effect on adenocarcinoma of the colon. B7H3 is also expressed in pancreatic cancer and is associated with increased treatment efficacy. Pancreatic cancer patients with high tumor B7H3 levels have a significantly better postoperative prognosis than patients with low tumor B7H3 levels (Yang et al., IntJ Biol Sci 2020;

16(11): 1767-1773). Due to its selective expression on solid tumors and its pro-tumorigenic function, B7H3 is the target of several anti-cancer agents including enoblituzumab, omburtamab, MGD009, MGC018, DS-7300a, and CAR-T cells.

In one aspect, therefore, an anti-B7H3 polypeptide may be incorporated into an immunotherapeutic compound. Immunotherapeutic compounds can provide individualized treatment that activates or suppresses the immune system to amplify or diminish an immune response and is developing rapidly for treating various forms of cancer. Immunotherapy for cancer, such as chimeric antigen receptor (CAR)-T cells, CAR-natural killer (NK) cells, PD-1 and PD-L1 inhibitor, aims to help a subject's immune system fight cancer. The activation of T cells depends on both the specific combination of T cell receptor (TCR) and peptide-bound major histocompatibility complex (MHC), and the interplay of co-stimulatory molecules of T cell with ligands on antigen presenting cells (APCs). The B7 families, peripheral membrane proteins on activated APCs, have been shown to participate in regulation of T cell responses. Recent studies indicate that the upregulation of inhibitory B7 molecules in the cancer microenvironment is highly related to the immune evasion of tumors. As a newly identified member of the B7 family, B7H3 could promote the activation of T cells and the production of IFN-γ.

In one embodiment, this disclosure describes an anti-B7H3 polypeptide that includes at least one complementarity-determining region (CDR) as reflected in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6. Exemplary anti-B7H3 polypeptides that include each of these CDRs is reflected in the amino acid sequences of SEQ ID NO: 1, SEQ ID NO:2, and SEQ ID NO:3. As used herein, the terms “peptide,” “polypeptide,” and “protein” refer interchangeably to any chain of at least two amino acids, linked by a covalent chemical bound. Thus, as used herein, the term “polypeptide” can refer to the complete amino acid sequence coding for an entire protein or to a portion thereof. As used herein, the terms “anti-B7H3 peptide,” “anti-B7H3 protein,” “B7H3 binding peptide,” and “B7H3 targeting peptide” generally refer to any peptide or polypeptide (including a protein or a fusion protein) that can specifically bind to B7H3.

In some embodiments, the anti-B7H3 polypeptide can be an antibody or an antibody fragment that includes a CDR region. As used herein, “CDR region” refers to one or more of the three complementarity-determining regions (CDRs) of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3. The CDRs are identified separately as SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6. In some aspects, the polypeptide encodes the light chain and the heavy chain of a B7H3 targeting protein.

A “protein coding sequence” or a sequence that “encodes” a particular polypeptide is a nucleic acid sequence that is transcribed (in the case of DNA) and is translated (in the case of mRNA) into a polypeptide in vitro or in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxyl) terminus. A coding sequence can include, but is not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and even synthetic DNA sequences. A transcription termination sequence will usually be located 3' to the coding sequence.

The term “antibody” refers to a molecule that contains at least one antigen binding site that immunospecifically binds to a particular antigen target of interest. The term “antibody” thus includes but is not limited to a full length antibody and/or its variants, a fragment thereof, peptibodies and variants thereof, monoclonal antibodies (including full-length monoclonal antibodies), multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, human antibodies, humanized antibodies, and antibody mimetics that mimic the structure and/or function of an antibody or a specified fragment or portion thereof, including single chain antibodies and fragments thereof. Thus, as used herein, the term “antibody” encompasses antibody fragments capable of binding to a biological molecule (such as an antigen or receptor) or a portion thereof, including but not limited to Fab, Fab' and F(ab')₂, pFc', Fd, a single domain antibody (sdAb), a variable fragment (Fv), a single-chain variable fragment (scFv) or a disulfide-linked Fv (sdFv); a diabody or a bivalent diabody; a linear antibody; a single-chain antibody molecule; and a multispecific antibody (e.g., a tribody) formed from antibody fragments. The antibody can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgAl and IgA2), or subclass.

The anti-B7H3 protein described herein can be any protein that selectively binds to B7H3. Exemplary anti-B7H3 proteins include SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and functional variants thereof. As used herein, a protein is a “functional variant” to a reference protein if the amino acid sequence of the protein possesses a specified amount of identity compared to the reference protein and retains the activity of the reference protein. Structural similarity of two proteins can be determined by aligning the residues of the two proteins (for example, a candidate protein and the protein of, for example, SEQ ID NO: 1 SEQ ID NO:2, or SEQ ID NO:3) to optimize the number of identical amino acids along the lengths of their sequences; gaps in either or both sequences are permitted in making the alignment in order to optimize the number of identical amino acids, although the amino acids in each sequence must nonetheless remain in their proper order. A candidate protein is the protein being compared to the reference protein (e.g., SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3). A candidate protein can be isolated, for example, from an animal, or can be produced using recombinant techniques, or chemically or enzymatically synthesized.

A pair-wise comparison analysis of amino acid sequences can be carried out using, for example, the BESTFIT algorithm in the GCG package (version 10.2, Madison WI).

Alternatively, proteins may be compared using the Blastp program of the BLAST 2 search algorithm, as described by Tatiana et al., (FEMS Microbiol Lett, 174, 247-250 (1999)), and available on the National Center for Biotechnology Information (NCBI) website. The default values for all BLAST 2 search parameters may be used, including matrix = BLOSUM62; open gap penalty = 11, extension gap penalty = 1, gap x dropoff = 50, expect = 10, wordsize = 3, and filter on.

In the comparison of two amino acid sequences, structural similarity may be referred to by percent “identity” or may be referred to by percent “similarity.” “Identity” refers to the presence of identical amino acids. “Similarity” refers to the presence of not only identical amino acids but also the presence of conservative substitutions. A conservative substitution for an amino acid in an anti-B7H3 protein may be selected from other members of the class to which the amino acid belongs. For example, it is well-known in the art of protein biochemistry that an amino acid belonging to a grouping of amino acids having a particular size or characteristic (such as charge, hydrophobicity and hydrophilicity) can be substituted for another amino acid without altering the activity of a protein, particularly in regions of the protein that are not directly associated with biological activity. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and tyrosine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. The positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Conservative substitutions include, for example, Lys for Arg and vice versa to maintain a positive charge; Glu for Asp and vice versa to maintain a negative charge; Ser for Thr so that a free -OH is maintained; and Gin for Asn to maintain a free -ML. Likewise, biologically active analogs of a protein containing deletions or additions of one or more contiguous or noncontiguous amino acids that do not eliminate a functional activity of the protein are also contemplated.

Generally, portions of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 outside of the CDRs are more amenable to variation while maintaining anti-B7H3 functionality — i.e., specifically bind to B7H3. Thus, an anti-B7H3 protein can include one, two, or all three of the CDRs of SEQ ID NO: 1, SEQ ID NO:2, and SEQ ID NO:3-i.e., the amino acids of SEQ ID NO:4 (CDR1), SEQ ID NO:5 (CDR2), and SEQ ID NO:6 (CDR3).

An anti-B7H3 protein as described herein can include a protein with at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least

88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least

95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence similarity to SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3.

95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3.

Variants of the disclosed sequences also include proteins, or full-length protein, that contain substitutions, deletions, or insertions into the protein backbone, that would still leave at least about 70% homology to the original protein over the corresponding portion. A yet greater degree of departure from homology is allowed if like-amino acids, i.e., conservative amino acid substitutions, do not count as a change in the sequence. Examples of conservative substitutions involve amino acids that have the same or similar properties. Illustrative amino acid conservative substitutions include the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine, glutamine, or glutamate; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; valine to isoleucine to leucine.

In some aspects, an anti-B7H3 protein can include additional sequences, such as, for example, amino acids appended to the C-terminal or N-terminal of the anti-B7H3 protein. Such modifications can, for example, facilitate purification by trapping on columns, the use of antibodies, or facilitate recovery when expressed recombinantly in a microbe. Such tags include, for example, a histidine-rich tag (e.g., SEQ ID NO:8) that allows purification of proteins on nickel columns and/or a leader sequence (e.g., SEQ ID NO:7) that can traffic recombinantly- expressed protein to the membrane of the cell in which it is recombinantly expressed. Such gene modification techniques and suitable additional sequences are well known in the molecular biology arts. In some embodiments, the C-terminal and/or N-terminal modification may be cleaved from the anti-B7H3 protein before being incorporated into, for example, a pharmaceutical composition. In other embodiments, retaining a C-terminal or N-terminal modification may be desired for a given application — i.e., to facilitate immobilization to a substrate.

In another aspect, this disclosure describes a multispecific compound that includes a targeting domain that includes an anti-B7H3 protein. The anti-B7H3 protein includes at least one CDR from SEQ ID NO:1. SEQ ID NO:2, or SEQ ID NO:3 such as, for example, the amino acid sequence of SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6. The multispecific compound further includes an immune cell engaging domain operably linked to the targeting domain.

The terms “multispecific compound” and “multispecific protein” refer to a “fusion molecule” or “fusion protein” and refer to a biologically active polypeptide, including two or more binding domains, with or without a further effector molecule, covalently linked (e.g., fused) by recombinant, chemical, or other suitable method. For example, a binding domain can be linked to another binding domain through a peptide linker sequence. Alternatively, a peptide linker may be used to assist in the construction of the fusion molecule.

As used herein, the term “operably linked” refers to a direct or indirect covalent linking between the domains of the multispecific compound. Thus, two domains that are operably linked may be directly covalently coupled to one another. Conversely, two operably linked domains may be connected by mutual covalent linking to an intervening moiety (e.g., a flanking sequence or linker). Two domains may be considered operably linked if, for example, they are separated by the third domain, with or without one or more intervening flanking sequences.

Domains of a multispecific compound can be in assembled operable linkage with one another using one or more linkers. The term “linker” as used herein refers any bond, small molecule, peptide sequence, or other vehicle that physically links the domains. Linkers can be susceptible to or be substantially resistant to acid-induced cleavage, light-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage and disulfide bond cleavage at conditions under which the compound or the antibody remains active. Linkers are classified upon their chemical motifs, well known in the art, including disulfide groups, hydrazine or peptides (cleavable), or thioester groups (non-cleavable). Linkers also include charged linkers, and hydrophilic forms thereof as known in the art.

Suitable linker for linking domains of a multispecific anti-B7H3 compound can include natural linkers, empirical linkers, or a combination of natural and empirical linkers. Natural linkers are derived from multi-domain proteins, which are naturally present between protein domains. Properties of natural linkers such as, for example, length, hydrophobicity, amino acid residues, and/or secondary structure can be exploited to confer desirable properties to a multi- domain compound that includes natural linkers connecting functional domains.

The studies of linkers in natural multi-domain proteins have led to the generation of many empirical linkers with various sequences and conformations for the construction of recombinant fusion proteins. Empirical linkers can be classified in three types: flexible linkers, rigid linkers, and cleavable linkers. Flexible linkers can provide a certain degree of movement or interaction at the joined domains. Flexible linkers typically include small, non-polar (e.g., Gly) or polar (e.g., Ser or Thr) amino acids, which provide flexibility, and allow for mobility of the connected functional domains. Rigid linkers can successfully keep a fixed distance between domains to maintain their independent functions, which can provide efficient separation of the protein domains and/or sufficiently reduce interference between functional domains. Cleavable linkers can allow one to control release of functional domains in vivo. By taking advantage of unique in vivo processes, cleavable linkers can be cleaved under specific conditions such as the presence of reducing reagents or proteases. This type of linker can reduce steric hindrance, improve bioactivity, and/or achieve independent actions/metabolism of individual domains of recombinant fusion proteins after linker cleavage.

Exemplary linkers are reflected in the amino acid sequences of SEQ ID NOs: 12-18.

In one exemplary application, an anti-B7H3 polypeptide can be incorporated into a multispecific NK engager compound that includes, at a minimum, the anti-B7H3 protein and an NK engager domain.

Natural killer (NK) cells are cytotoxic lymphocytes of the innate immune system capable of immune surveillance. Like T cells, NK cells deliver a store of membrane penetrating and apoptosis-inducing granzyme and perforin granules. Unlike T cells, NK cells do not require antigen priming and recognize targets by engaging activating receptors in the absence of MHC recognition.

NK cells express CD16, an activation receptor that binds to the Fc portion of IgG antibodies and is involved in antibody-dependent cell-mediated cytotoxicity (ADCC). NK cells are regulated by IL-15, which can induce increased antigen-dependent cytotoxicity, lymphokine- activated killer activity, and/or mediate interferon (IFN), tumor-necrosis factor (TNF) and/or granulocyte-macrophage colony-stimulating factor (GM-CSF) responses. IL-15 can also drive NK cell proliferation and survival, thus enhancing expansion and persistence of NK cells. All of these IL-15 -activated functions contribute to improved cancer defense.

Therapeutically, adoptive transfer of NK cells can, for example, induce remission in subjects with refractory acute myeloid leukemia (AML) when combined with lymphodepleting chemotherapy and IL-2 to stimulate survival and in vivo expansion of NK cells. This therapy can be limited by lack of antigen specificity and IL-2-mediated induction of regulatory T (Treg) cells that suppress NK cell proliferation and function. Generating a reagent that drives NK cell antigen specificity, expansion, and/or persistence, while bypassing the negative effects of Treg inhibition, can enhance NK-cell-based immunotherapies.

In one aspect, therefore, this disclosure describes the design, construction, and use of a trispecific molecule that includes two domains capable of driving NK-cell-mediated killing of B7H3⁺ tumor cells and an intramolecular NK activating domain capable of generating an NK cell self-sustaining signal. The trispecific molecule can drive NK cell proliferation and/or enhance NK-cell-driven cytotoxicity against, for example, B7H3⁺ cancer cells or B7H3⁺ cancer cell-derived cell lines.

This disclosure describes, in one aspect, trispecific killer engager molecules that generally include a targeting domain that selectively targets B7H3, an NK cell engager domain (e.g., CD 16, CD16+CD2, CD16+DNAM, NKp46, CD16+NKp46, NKG2D, NK2C), and anNK activating domains (e.g., IL-15, IL-12, IL-18, IL-21, or other NK cell enhancing cytokine, chemokine, and/or activating molecule), with each domain operably linked to the other domains. As used herein, the terms “selectively targets” and “selectively binds” refer to the ability to differentiate between two or more alternatives such as, for example, having differential affinity, to any degree, for a particular target. Particularly in the context of a trispecific compound, the term “operably linked” includes domains connected by mutual covalent linking to an intervening moiety (e.g., a flanking sequence, multiple flanking sequences, and/or another functional domain). Thus, two domains may be considered operably linked if, for example, they are separated by a third functional domain, with or without one or more intervening flanking sequences.

The targeting domain can include any moiety that selectively binds to a B7H3⁺ target such as, for example, a tumor cell, a target in the cancer stroma, or an immobilized B7H3⁺ cell. Thus, a targeting domain can include, for example, an anti-B7H3 antibody. In some embodiments, an anti-B7H3 antibody can include an anti-B7H3 polypeptide as described in detail herein. In one exemplary embodiment, the anti-B7H3 polypeptide can include one or more of the complementarity-determining regions (CDRs) of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3. In some embodiments, the anti-B7H3 protein can include two or all three of the CDRs of SEQ ID NO: 1, SEQ ID NO:2, and SEQ ID NO:3. In some embodiments, the anti-B7H3 protein can include SEQ ID NO: 1, SEQ ID NO:2, or a variant thereof (e.g., SEQ ID NO:3). Suitable alternative variants are described herein.

The NK engaging domain can include any moiety that binds to and/or activates an NK cell and/or any moiety that blocks inhibition of an NK cell. In some embodiments, the NK engaging domain can include an antibody that selectively binds to a component of the surface of an NK cell. In other embodiments, the NK engaging domain can include a ligand or small molecule that selectively binds to a component of the surface of an NK cell. Thus, for brevity, reference to an antibody that selectively binds to a component of the surface of an NK cell includes any antibody fragment that exhibits the described binding character. Similarly, reference to a ligand that selectively binds to a component of the surface of an NK cell includes any fragment of the ligand that exhibits the described binding character.

In some embodiments, the NK engaging domain can selectively bind to a receptor at least partially located at the surface of an NK cell. In certain embodiments, the NK engaging domain can serve a function of binding an NK cell and thereby bring the NK cell into spatial proximity with a target to which the targeting domain selectively binds. In certain embodiments, however, the NK engaging domain can selectively bind to a receptor that activates the NK cell and, therefore, also possess an activating function. As described above, activation of the CD 16 receptor can elicit antibody-dependent cell-mediated cytotoxicity. Thus, in certain embodiments, the NK engaging domain can include at least a portion of an anti-CD 16 receptor antibody effective to selectively bind to the CD 16 receptor. In other embodiments, the NK engager cell domain may interrupt mechanisms that inhibit NK cells. In such embodiments, the NK engager domain can include, for example, anti-PDl/PDLl, anti-NKG2A, anti-TIGIT, anti -killer- immunoglobulin receptor (KIR), and/or any other inhibition blocking domain.

One can design the NK engaging domain to possess a desired degree of NK selectivity and, therefore, a desired immune engaging character. For example, CD 16 has been identified as Fc receptors FcγRIIIa (CD 16a) and FcγRIIIb (CD 16b). These receptors bind to the Fc portion of IgG antibodies that then activates the NK cell for antibody-dependent cell-mediated cytotoxicity. Anti-CD 16 antibodies selectively bind to NK cells, but also can bind to neutrophils. Anti-CD 16a antibodies selectively bind to NK cells, but do not bind to neutrophils. A trispecific killer engager compound that includes an NK engaging domain that includes an anti-CD 16a antibody can bind to NK cells but not bind to neutrophils. Thus, in circumstances where one may want to engage NK cells but not engage neutrophils, one can design the NK engaging domain of the trispecific killer engager compound to include an anti-CD 16a antibody.

In some embodiments, the NK cell engaging domain can involve the use of a humanized CD 16 engager derived from an animal nanobody. While an scFv has a heavy variable chain component and a light variable chain component joined by a linker, a nanobody consists of a single monomeric variable chain—i.e., a variable heavy chin or a variable light chain — that is capable of specifically engaging a target. A single domain antibody (sdAb) may be derived from an antibody of any suitable animal such as, for example, a camelid (e.g., a llama or camel) or a cartilaginous fish. A single domain antibody can provide superior physical stability, an ability to bind deep grooves, and increased production yields compared to larger antibody fragments.

In one exemplary embodiment, an sdAb-based NK engager molecule can involve a humanized CD16 nanobody derived from a llama nanobody (GeneBank sequence EF561291; Behar et al., 2008. Protein Eng Des Sel. 21(1): 1-10), termed EF91. Upon confirming functionality of the molecule, the CDRs were cloned into a humanized camelid scaffold (Vincke et al., 2009. J Biol Chem. 284(5):3273-3284) to humanize the CD16 engager (SEQ ID NO: 19). The use of a humanized camelid sdAb in the NK engaging domain of a trispecific killer engager compound can increase drug yield, increase stability, and/or increase NK-cell-mediated antibody-dependent cellular cytotoxicity (ADCC) efficacy. While described herein in the context of various embodiments in which the NK engaging domain includes an anti-CD 16 sdAb or anti-CD 16 scFv, the NK engaging domain can include any antibody or other ligand that selectively binds to CD 16. Moreover, the NK engaging domain can include an antibody or ligand that selectively binds to any NK cell receptor such as, for example, the cell cytotoxicity receptor 2B4, low affinity Fc receptor CD 16, killer immunoglobulin like receptors (KIR), CD2, NKG2A, TIGIT, NKG2C, LIR-1, and/or DNAM-1.

In one aspect, the immune cell is a T cell or a natural killer (NK) cell. In another aspect, the immune cell is an NK cell; and the immune cell engaging domain includes a ligand or antibody that specifically binds to CD 16. In some aspects, the antibody that specifically binds to CD16 includes a an scFv, a F(ab)₂, a Fab, or a single domain antibody.

As explained in more detail above, “antibody” refers generally an immunoglobulin or a fragment thereof and thus encompasses a monoclonal antibody, a fragment thereof (e.g., scFv, Fab, F(ab')₂, Fv, sdAb, or other modified form of an antibody, including a humanized form of an antibody or fragment thereof). Thus, for brevity, reference to an antibody that selectively binds to B7H3 includes any antibody or antibody fragment that exhibits the described binding character. Similarly, reference to an antibody that selectively binds to CD 16 (or any other NK cell receptor) includes any antibody or antibody fragment that exhibits the described binding character. In some aspects, the immune cell engaging domain includes a ligand or antibody that specifically binds to CD 16 such as, for example, an antibody fragment having the amino acid sequence set forth in SEQ ID NO: 19.

In some embodiments, the NK engaging domain and the targeting domain can be linked using any one of the linkers reflected in SEQ ID NOs: 12-18. In particular embodiments, a bispecific anti-B7H3 compound can include the amino acid sequence of SEQ ID NO:20 or SEQ ID NO:21. In some embodiments, a bispecific anti-B7H3 compound can include the amino acid sequence of SEQ ID NO:20 or SEQ ID NO:21 with deletion of the leader sequence, a deletion of the VDE linker and HID tag, or a deletion of both the leader sequence and the VDE linker and HIS tag.

In another aspect, the multispecific anti-B7H3 compound further includes an immune cell activating domain. In some embodiments, the immune cell can be an NK cell and the immune cell activating domain includes a NK activating cytokine or a functional portion thereof. The NK activating domain can include a “immune cell activating domain”, e.g., an amino acid sequence that activates NK cells, promotes sustaining NK cells, or otherwise promotes NK cell activity. For example, NK cells are responsive to a variety of cytokines including, but not limited to, IL-15, which is involved in NK cell homeostasis, proliferation, survival, activation, and/or development. IL-15 and IL-2 share several signaling components, including the IL-2/IL- 15Rβ (CD122) and the common gamma chain (CD132). Unlike IL-2, IL-15 does not stimulate Tregs, allowing for NK cell activation while bypassing Treg inhibition of the immune response. Besides promoting NK cell homeostasis and proliferation, IL-15 can rescue NK cell functional defects that can occur in the post-transplant setting. IL-15 also can stimulate CD8⁺ T cell function, further enhancing its immunotherapeutic potential. In addition, based on pre-clinical studies, toxicity profiles of IL-15 may be more favorable than IL-2 at low doses.

Therefore, the NK activating domain can be, or can be derived from, one or more cytokines that can activate and/or sustain NK cells. As used herein, the term “derived from” refers to an amino acid fragment of a cytokine (e.g., IL-15) that is sufficient to provide NK cell activating and/or sustaining activity. In embodiments that include more than one NK activating domain, the NK activating domains may be provided in series or in any other combination. Additionally, each cytokine-based NK activating domain can include either the full amino acid sequence of the cytokine or may be an amino acid fragment, independent of the nature of other NK activating domains included in the trispecific killer engager compound. Exemplary cytokines on which an NK activating domain may be based include, for example, IL-15, IL-18, IL-12, and IL-21. Thus, while described in detail herein in the context of an exemplary model embodiment in which the NK activating domain is derived from IL-15, a trispecific killer engager compound may be designed using an NK activating domain that is, or is derived from, any suitable cytokine.

For brevity in this description, reference to an NK activating domain by identifying the cytokine on which it is based includes both the full amino acid sequence of the cytokine, any suitable amino acid fragment of the cytokine, and or a modified version of the cytokine that includes one or more amino acid substitutions. Thus, reference to an “IL-15” NK activating domain includes an NK activating domain that includes the full amino acid sequence of IL-15, an NK activating domain that includes a fragment of IL-15 (e.g., SEQ ID NO: 11), a functional variant thereof, or an NK activating domain that includes an amino acid substitution compared to the wild-type IL-15 amino acid sequence. For example, an NK activating domain can include a fragment of IL-15 that includes an N-to-D or an N-to-A amino acid substitution at position 72 of SEQ ID NO: 11. Reference to position 72 of SEQ ID NO: 11 merely refers to the location of the amino acid substitution regardless of the particular fragment of IL-15 that may be used as the NK activating domain. Thus, the NK activating domain can include a fragment of IL-15 other than the fragment reflected in SEQ ID NO: 11 and that fragment can have an N-to-D or an N-to-A amino acid substitution at the position of the alternative IL-15 fragment that corresponds to position 72 of SEQ ID NO: 11.

In some embodiments, trispecific anti-B7H3 compounds include domains linked by one or more of the linkers reflected in SEQ ID NOs: 12-18 or a combination of the linkers reflected in SEQ ID NOs: 12-18. In specific exemplary embodiments, the NK engaging domain is linked to the NK activating domain using the linker of SEQ ID NO: 14, while the NK activating domain is linked to the targeting domain using the linker of SEQ ID NO:15 (e.g., SEQ ID NOs:22-25).

In another aspect, this disclosure describes an isolated nucleic acid sequence that encodes any embodiment of an anti-B7H3 compound, one of the compounds described herein. In some embodiments, the isolated nucleic is the nucleic acid sequence of any one of SEQ ID NOs: 26- 33. Given the amino acid sequence of any anti-B7H3 polypeptide, or multispecific anti-B7H3 compound that includes an anti-B7H3 polypeptide, a person of ordinary skill in the art can determine the full scope of polynucleotides that encode that amino acid sequence using conventional, routine methods.

As used herein, the term “nucleic acid” or “oligonucleotide” refers to polynucleotides such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Nucleic acids include but are not limited to genomic DNA, cDNA, mRNA, iRNA, miRNA, tRNA, ncRNA, rRNA, and recombinantly produced and chemically synthesized molecules such as aptamers, plasmids, anti- sense DNA strands, shRNA, ribozymes, nucleic acids conjugates, and oligonucleotides. A nucleic acid may be single-stranded, double-stranded, linear, or covalently circularly closed molecule. A nucleic acid can be isolated. The term “isolated nucleic acid” means, that the nucleic acid (i) was amplified in vitro, for example via polymerase chain reaction (PCR), (ii) was produced recombinantly by cloning, (iii) was purified, for example, by cleavage and separation by gel electrophoresis, (iv) was synthesized, for example, by chemical synthesis, or (vi) extracted from a sample. A nucleic might be introduced — i.e., transfected — into cells. When RNA is used to transfect cells, the RNA may be modified by stabilizing modifications, capping, or polyadenylation.

As used herein “amplified DNA” or “PCR product” refers to an amplified fragment of DNA of defined size. Various techniques are available and well known in the art to detect PCR products. PCR product detection methods include, but are not restricted to, gel electrophoresis using agarose or polyacrylamide gel and adding ethidium bromide staining (a DNA intercalant), labeled probes (radioactive or non-radioactive labels, southern blotting), labeled deoxyribonucleotides (for the direct incorporation of radioactive or non-radioactive labels) or silver staining for the direct visualization of the amplified PCR products; restriction endonuclease digestion, that relies agarose or polyacrylamide gel or high-performance liquid chromatography (HPLC); dot blots, using the hybridization of the amplified DNA on specific labeled probes (radioactive or non-radioactive labels); high-pressure liquid chromatography using ultraviolet detection; electro-chemiluminescence coupled with voltage-initiated chemical reaction/photon detection; and direct sequencing using radioactive or fluorescently labeled deoxyribonucleotides for the determination of the precise order of nucleotides with a DNA fragment of interest, oligo ligation assay (OLA), PCR, qPCR, DNA sequencing, fluorescence, gel electrophoresis, magnetic beads, allele specific primer extension (ASPE) and/or direct hybridization.

Generally, nucleic acid can be extracted, isolated, amplified, or analyzed by a variety of techniques such as those described by Green and Sambrook, Molecular Cloning: A Laboratory Manual (Fourth Edition), Cold Spring Harbor Laboratory Press, Woodbury, NY 2,028 pages (2012); or as described in U.S. Pat. 7,957,913; U.S. Pat. 7,776,616; U.S. Pat. 5,234,809; U.S.

Pub. 2010/0285578; and U.S. Pub. 2002/0190663. Examples of nucleic acid analysis include, but are not limited to, sequencing and DNA-protein interaction. Sequencing may be by any method known in the art. DNA sequencing techniques include classic dideoxy sequencing reactions (Sanger method) using labeled terminators or primers and gel separation in slab or capillary, and next generation sequencing methods such as sequencing by synthesis using reversibly terminated labeled nucleotides, pyrosequencing, 454 sequencing, Illumina/Solexa sequencing, allele specific hybridization to a library of labeled oligonucleotide probes, sequencing by synthesis using allele specific hybridization to a library of labeled clones that is followed by ligation, real time monitoring of the incorporation of labeled nucleotides during a polymerization step, polony sequencing, and SOLiD sequencing. Separated molecules may be sequenced by sequential or single extension reactions using polymerases or ligases as well as by single or sequential differential hybridizations with libraries of probes.

In another aspect, this disclosure describes proteins encoded by any of the nucleic acid sequences described herein. In some embodiments, the protein can have an amino acid sequence that includes the amino acid sequence of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, or any protein having 90% or greater amino acid identity thereto.

In another aspect, this disclosure describes a host cell including any of the isolated nucleic acid sequences and/or proteins described herein.

The nucleic acid constructs of the present invention may be introduced into a host cell to be altered, thus allowing expression of the chimeric protein within the cell, thereby generating a genetically engineered cell. A variety of methods are known in the art and suitable for introduction of nucleic acid into a cell, including viral and non-viral mediated techniques. Examples of typical non-viral mediated techniques include, but are not limited to, electroporation, calcium phosphate mediated transfer, nucleofection, sonoporation, heat shock, magnetofection, liposome mediated transfer, microinjection, microprojectile mediated transfer (nanoparticles), cationic polymer mediated transfer (DEAE-dextran, polyethylenimine, polyethylene glycol (PEG) and the like) or cell fusion. Other methods of transfection include proprietary transfection reagents such as LIPOFECTAMINE (Thermo Fisher Scientific, Inc., Waltham, MA), HP.UMAC (Dojindo Molecular Technologies, Inc., Rockville, MD), FUGENE (Promega Corp., Madison, WI), JETPEI (Polyplus Transfection, Illkirch, France), EFFECTENE (Qiagen, Hilden, Germany) and DreamFect (OZ Biosciences, Inc USA, San Diego, CA).

The nucleic acid constructs described herein may be introduced into a host cell to be altered, thus allowing expression within the cell of the protein encoded by the nucleic acid. A variety of host cells are known in the art and suitable for protein expression. Examples of typical cell used for transfection and protein expression include, but are not limited to, a bacterial cell, a eukaryotic cell, a yeast cell, an insect cell, or a plant cell such as, for example, E. coli , Bacillus , Streptomyces, Pichia pastoris , Salmonella typhimurium , Drosophila S2, Spodoptera SJ9, CHO, COS (e g., COS-7), 3T3-F442A, HeLa, HUVEC, HUAEC, NIH 3T3, Jurkat, 293, 293H, or 293F.

In some aspects, the host cell is a T cell, an NK cell, or a macrophage. In a further aspect, this disclosure describes a pharmaceutical composition that includes any one of the multispecific anti-B7H3 compounds described herein and a pharmaceutically acceptable carrier.

A multispecific anti-B7H3 compound, such as a trispecific killer engager compound as described herein may be formulated with a pharmaceutically acceptable carrier. As used herein, “carrier” includes any solvent, dispersion medium, vehicle, coating, diluent, antibacterial, and/or antifungal agent, isotonic agent, absorption delaying agent, buffer, carrier solution, suspension, colloid, and the like. The use of such media and/or agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients also can be incorporated into the compositions. As used herein, “pharmaceutically acceptable” refers to a material that is not biologically or otherwise undesirable, i.e., the material may be administered to an individual along with a trispecific killer engager compound without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.

A multispecific compound, such as a trispecific killer engager compound may therefore be formulated into a pharmaceutical composition. The pharmaceutical composition may be formulated in a variety of forms adapted to a preferred route of administration. Thus, a composition can be administered via known routes including, for example, oral, parenteral (e.g., intradermal, transcutaneous, subcutaneous, intramuscular, intravenous, intraperitoneal, etc.), or topical (e.g., intranasal, intrapulmonary, intramammary, intravaginal, intrauterine, intradermal, transcutaneous, rectally, etc.). A pharmaceutical composition can be administered to a mucosal surface, such as by administration to, for example, the nasal or respiratory mucosa (e.g., by spray or aerosol). A composition also can be administered via a sustained or delayed release.

Thus, a multispecific compound, such as a trispecific killer engager compound may be provided in any suitable form including but not limited to a solution, a suspension, an emulsion, a spray, an aerosol, or any form of mixture. The composition may be delivered in formulation with any pharmaceutically acceptable excipient, carrier, or vehicle. For example, the formulation may be delivered in a conventional topical dosage form such as, for example, a cream, an ointment, an aerosol formulation, a non-aerosol spray, a gel, a lotion, and the like. The formulation may further include one or more additives including such as, for example, an adjuvant, a skin penetration enhancer, a colorant, a fragrance, a flavoring, a moisturizer, a thickener, and the like.

A formulation may be conveniently presented in unit dosage form and may be prepared by methods well known in the art of pharmacy. Methods of preparing a composition with a pharmaceutically acceptable carrier include the step of bringing a multispecific compound or trispecific killer engager compound into association with a carrier that constitutes one or more accessory ingredients. In general, a formulation may be prepared by uniformly and/or intimately bringing the active molecule into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into the desired formulations.

The amount of multispecific compound or trispecific killer engager compound administered can vary depending on various factors including, but not limited to, the specific trispecific killer engager compound being used, the weight, physical condition, and/or age of the subject, and/or the route of administration. Thus, the absolute weight of trispecific killer engager compound included in a given unit dosage form can vary widely, and depends upon factors such as the species, age, weight and physical condition of the subject, and/or the method of administration. Accordingly, it is not practical to set forth generally the amount that constitutes an amount of multispecific compound or trispecific killer engager compound effective for all possible applications. Those of ordinary skill in the art, however, can readily determine the appropriate amount with due consideration of such factors.

In some embodiments, the method can include administering sufficient multispecific compound or trispecific killer engager compound to provide a dose of, for example, from about 100 ng/kg to about 50 mg/kg to the subject, although in some embodiments the methods may be performed by administering the multispecific compound or trispecific killer engager compound in a dose outside this range. In some of these embodiments, the method includes administering sufficient the multispecific compound or trispecific killer engager compound to provide a dose of from about 10 μg/kg to about 5 mg/kg to the subject, for example, a dose of from about 100 μg/kg to about 1 mg/kg.

Alternatively, the dose may be calculated using actual body weight obtained just prior to the beginning of a treatment course. For the dosages calculated in this way, body surface area (m²) is calculated prior to the beginning of the treatment course using the Dubois method: m² = (wt kg⁰⁴²⁵ x height cm⁰⁷²⁵) x 0.007184.

In some embodiments, the method can include administering sufficient the multispecific compound or trispecific killer engager compound to provide a dose of, for example, from about 0.01 mg/m² to about 10 mg/m².

In another aspect, this disclosure describes a method including administering to a subject a multispecific compound in an amount effective to induce NK-mediated killing of a cell, the multispecific compound including a targeting domain including one of the anti-B7H3 proteins described herein; and an NK engaging domain operably linked to the anti-B7H3 protein.

In another aspect, this disclosure describes a method for stimulating expansion of NK cells in vivo including administering to a subject an effective amount of multispecific compound including a targeting domain including one of the anti-B7H3 proteins described herein; and an NK engaging domain operably linked to the anti-B7H3 protein.

In another aspect, this disclosure describes methods of killing a target cell in a subject. Generally, the method includes administering to the subject an anti-B7H3 multispecific compound in an amount effective to induce NK-mediated killing of the target cells. “Treat” or variations thereof refer to reducing, limiting progression, ameliorating, or resolving, to any extent, the symptoms or signs related to a condition. As used herein, “ameliorate” refers to any reduction in the extent, severity, frequency, and/or likelihood of a symptom or clinical sign characteristic of a particular condition; “symptom” refers to any subjective evidence of disease or of a subject's condition; and “sign” or “clinical sign” refers to an objective physical finding relating to a particular condition capable of being found by one other than the subject.

A “treatment” may be therapeutic or prophylactic. “Therapeutic” and variations thereof refer to a treatment that ameliorates one or more existing symptoms or clinical signs associated with a condition. “Prophylactic” and variations thereof refer to a treatment that limits, to any extent, the development and/or appearance of a symptom or clinical sign of a condition. Generally, a “therapeutic” treatment is initiated after the condition manifests in a subject, while “prophylactic” treatment is initiated before a condition manifests in a subject. Thus, in certain embodiments, the method can involve prophylactic treatment of a subject at risk of developing a condition. “At risk” refers to a subject that may or may not actually possess the described risk. Thus, for example, a subject “at risk” for developing a specified condition is a subject that possesses one or more indicia of increased risk of having, or developing, the specified condition compared to individuals who lack the one or more indicia, regardless of the whether the subject manifests any symptom or clinical sign of having or developing the condition. Exemplary indicia of a condition can include, for example, genetic predisposition, ancestry, age, sex, geographical location, lifestyle, or medical history. Treatment may also be continued after symptoms have resolved, for example to prevent or delay their recurrence.

In some cases, the treatment can involve administering the anti-B7H3 multispecific compound to a subject so that the anti-B7H3 multispecific compound can stimulate endogenous NK cells in vivo. Using an anti-B7H3 multispecific compound as a part of an in vivo can make NK cells antigen specific with simultaneous co-stimulation, enhancement of survival, and expansion, which may be antigen specific. In other cases, the anti-B7H3 multispecific compound can be used in vitro as an adjuvant to NK cell adoptive transfer therapy. The terms “administration of’ and or “administering” should be understood to mean providing a pharmaceutical composition in a therapeutically effective amount to the subject in need of treatment. Administration routes can be enteral, topical, or parenteral. As such, administration routes include but are not limited to intracutaneous, subcutaneous, intravenous, intraperitoneal, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, transdermal, transtracheal, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, intrasternal, oral, sublingual buccal, rectal, vaginal, nasal ocular administrations, as well infusion, inhalation, and nebulization. The phrases “parenteral administration” and “administered parenterally” as used herein means modes of administration other than enteral and topical administration.

Accordingly, an anti-B7H3 multispecific compound may be administered before, during, or after the subject first exhibits a symptom or clinical sign of the condition. Treatment initiated before the subject first exhibits a symptom or clinical sign associated with the condition may result in decreasing the likelihood that the subject experiences clinical evidence of the condition compared to a subject to which the anti-B7H3 multispecific compound is not administered, decreasing the severity of symptoms and/or clinical signs of the condition, and/or completely resolving the condition. Treatment initiated after the subject first exhibits a symptom or clinical sign associated with the condition may result in decreasing the severity of symptoms and/or clinical signs of the condition compared to a subject to which the composition is not administered, and/or completely resolving the condition. The anti-B7H3 multispecific compound can be any embodiment of the anti-B7H3 multispecific compound described above having a targeting domain that selectively binds to an appropriate target cell population. In some cases, the target cell can include a tumor cell so that the method can involve treating cancer associated with the tumor cells. Thus, in some embodiments, the method can include ameliorating at least one symptom or clinical sign of the tumor.

In embodiments in which the target cell includes a tumor cell, the method can further include surgically resecting the tumor and/or reducing the size of the tumor through chemical (e.g., chemotherapeutic) and/or radiation therapy. Exemplary tumors that may be treated include tumors associated with prostate cancer, lung cancer, colon cancer, rectum cancer, urinary bladder cancer, melanoma, kidney cancer, renal cancer, oral cavity cancer, pharynx cancer, pancreas cancer, uterine cancer, thyroid cancer, skin cancer, head and neck cancer, cervical cancer, ovarian cancer and/or hematopoietic cancer.

Thus, in some embodiments, treating a subject includes a subject having, or at risk of having cancer. Generally, the method includes administering to the subject an effective amount of multispecific compound including a targeting domain including one of the anti-B7H3 proteins described herein and an NK engaging domain operably linked to the anti-B7H3 protein. As used herein, the term “cancer” refers to a group of diseases characterized by abnormal and uncontrolled cell proliferation starting at one site (primary site) with the potential to invade and to spread to other sites (secondary sites, metastases) which differentiates cancer (malignant tumor) from benign tumor. Virtually any organ can be affected, meaning more than 100 types of cancer can affect humans. Cancers can result from many causes including genetic predisposition, viral infection, exposure to ionizing radiation, exposure to an environmental pollutant, tobacco and/or alcohol use, obesity, poor diet, lack of physical activity, or any combination thereof. As used herein, “neoplasm” or “tumor” (and grammatical variations thereof) means new and abnormal growth of tissue, which may be benign or cancerous. In a related aspect, the neoplasm is indicative of a neoplastic disease or disorder, including but not limited, to various cancers. For example, such cancers can include prostate, pancreatic, biliary, colon, rectal, liver, kidney, lung, testicular, breast, ovarian, brain, and head and neck cancers, melanoma, sarcoma, multiple myeloma, leukemia, lymphoma, and the like. Exemplary cancers described by the national cancer institute include: Acute Lymphoblastic Leukemia, Adult; Acute Lymphoblastic Leukemia, Childhood; Acute Myeloid Leukemia, Adult; Adrenocortical Carcinoma; Adrenocortical Carcinoma, Childhood; AIDS- Related Lymphoma; AIDS-Related Malignancies; Anal Cancer; Astrocytoma, Childhood Cerebellar; Astrocytoma, Childhood Cerebral; Bile Duct Cancer, Extrahepatic; Bladder Cancer; Bladder Cancer, Childhood; Bone Cancer, Osteosarcoma/Malignant Fibrous Histiocytoma; Brain Stem Glioma, Childhood; Brain Tumor, Adult; Brain Tumor, Brain Stem Glioma, Childhood; Brain Tumor, Cerebellar Astrocytoma, Childhood; Brain Tumor, Cerebral Astrocytoma/Malignant Glioma, Childhood; Brain Tumor, Ependymoma, Childhood; Brain Tumor, Medulloblastoma, Childhood; Brain Tumor, Supratentorial Primitive Neuroectodermal Tumors, Childhood; Brain Tumor, Visual Pathway and Hypothalamic Glioma, Childhood; Brain Tumor, Childhood (Other); Breast Cancer; Breast Cancer and Pregnancy; Breast Cancer, Childhood; Breast Cancer, Male; Bronchial Adenomas/Carcinoids, Childhood: Carcinoid Tumor, Childhood; Carcinoid Tumor, Gastrointestinal; Carcinoma, Adrenocortical; Carcinoma, Islet Cell; Carcinoma of Unknown Primary; Central Nervous System Lymphoma, Primary; Cerebellar Astrocytoma, Childhood; Cerebral Astrocytoma/Malignant Glioma, Childhood; Cervical Cancer; Childhood Cancers; Chronic Lymphocytic Leukemia; Chronic Myelogenous Leukemia; Chronic Myeloproliferative Disorders; Clear Cell Sarcoma of Tendon Sheaths; Colon Cancer; Colorectal Cancer, Childhood; Cutaneous T-Cell Lymphoma; Endometrial Cancer; Ependymoma, Childhood; Epithelial Cancer, Ovarian; Esophageal Cancer; Esophageal Cancer, Childhood; Ewing's Family of Tumors; Extracranial Germ Cell Tumor, Childhood; Extragonadal Germ Cell Tumor; Extrahepatic Bile Duct Cancer; Eye Cancer, Intraocular Melanoma; Eye Cancer, Retinoblastoma; Gallbladder Cancer; Gastric (Stomach) Cancer; Gastric (Stomach) Cancer, Childhood; Gastrointestinal Carcinoid Tumor; Germ Cell Tumor, Extracranial, Childhood; Germ Cell Tumor, Extragonadal; Germ Cell Tumor, Ovarian; Gestational Trophoblastic Tumor; Glioma. Childhood Brain Stem; Glioma. Childhood Visual Pathway and Hypothalamic; Hairy Cell Leukemia; Head and Neck Cancer; Hepatocellular (Liver) Cancer, Adult (Primary); Hepatocellular (Liver) Cancer, Childhood (Primary); Hodgkin's Lymphoma, Adult; Hodgkin's Lymphoma, Childhood; Hodgkin's Lymphoma During Pregnancy; Hypopharyngeal Cancer; Hypothalamic and Visual Pathway Glioma, Childhood; Intraocular Melanoma; Islet Cell Carcinoma (Endocrine Pancreas); Kaposi's Sarcoma; Kidney Cancer; Laryngeal Cancer; Laryngeal Cancer, Childhood; Leukemia, Acute Lymphoblastic, Adult; Leukemia, Acute Lymphoblastic, Childhood; Leukemia, Acute Myeloid, Adult; Leukemia, Acute Myeloid, Childhood; Leukemia, Chronic Lymphocytic; Leukemia, Chronic Myelogenous; Leukemia, Hairy Cell; Lip and Oral Cavity Cancer; Liver Cancer, Adult (Primary); Liver Cancer,

Childhood (Primary); Lung Cancer, Non-Small Cell; Lung Cancer, Small Cell; Lymphoblastic Leukemia, Adult Acute; Lymphoblastic Leukemia, Childhood Acute; Lymphocytic Leukemia, Chronic; Lymphoma, AIDS — Related; Lymphoma, Central Nervous System (Primary); Lymphoma, Cutaneous T-Cell; Lymphoma, Hodgkin's, Adult; Lymphoma, Hodgkin's; Childhood; Lymphoma, Hodgkin's During Pregnancy; Lymphoma, Non-Hodgkin's, Adult; Lymphoma, Non-Hodgkin's, Childhood; Lymphoma, Non-Hodgkin's During Pregnancy; Lymphoma, Primary Central Nervous System; Macroglobulinemia, Waldenstrom's; Male Breast Cancer; Malignant Mesothelioma, Adult; Malignant Mesothelioma, Childhood; Malignant Thymoma; Medulloblastoma, Childhood; Melanoma; Melanoma, Intraocular; Merkel Cell Carcinoma; Mesothelioma, Malignant; Metastatic Squamous Neck Cancer with Occult Primary; Multiple Endocrine Neoplasia Syndrome, Childhood; Multiple Myeloma/Plasma Cell Neoplasm; Mycosis Fungoides; Myelodysplasia Syndromes; Myelogenous Leukemia, Chronic; Myeloid Leukemia, Childhood Acute; Myeloma, Multiple; Myeloproliferative Disorders, Chronic; Nasal Cavity and Paranasal Sinus Cancer; Nasopharyngeal Cancer; Nasopharyngeal Cancer,

Childhood; Neuroblastoma; Non-Hodgkin's Lymphoma, Adult; Non-Hodgkin's Lymphoma, Childhood; Non-Hodgkin's Lymphoma During Pregnancy; Non-Small Cell Lung Cancer; Oral Cancer, Childhood; Oral Cavity and Lip Cancer; Oropharyngeal Cancer; Osteosarcoma/Malignant Fibrous Histiocytoma of Bone; Ovarian Cancer, Childhood; Ovarian Epithelial Cancer; Ovarian Germ Cell Tumor; Ovarian Low Malignant Potential Tumor; Pancreatic Cancer; Pancreatic Cancer, Childhood, Pancreatic Cancer, Islet Cell; Paranasal Sinus and Nasal Cavity Cancer; Parathyroid Cancer; Penile Cancer; Pheochromocytoma; Pineal and Supratentorial Primitive Neuroectodermal Tumors, Childhood; Pituitary Tumor; Plasma Cell Neoplasm/Multiple Myeloma; Pleuropulmonary Blastoma; Pregnancy and Breast Cancer; Pregnancy and Hodgkin's Lymphoma; Pregnancy and Non-Hodgkin's Lymphoma; Primary Central Nervous System Lymphoma; Primary Liver Cancer, Adult; Primary Liver Cancer, Childhood; Prostate Cancer; Rectal Cancer; Renal Cell (Kidney) Cancer; Renal Cell Cancer, Childhood; Renal Pelvis and Ureter, Transitional Cell Cancer; Retinoblastoma; Rhabdomyosarcoma, Childhood; Salivary Gland Cancer; Salivary Gland's Cancer, Childhood; Sarcoma, Ewing's Family of Tumors; Sarcoma, Kaposi's; Sarcoma (Osteosarcoma) Malignant Fibrous Histiocytoma of Bone; Sarcoma, Rhabdomyosarcoma, Childhood; Sarcoma, Soft Tissue, Adult; Sarcoma, Soft Tissue, Childhood; Sezary Syndrome; Skin Cancer; Skin Cancer, Childhood; Skin Cancer (Melanoma); Skin Carcinoma, Merkel Cell; Small Cell Lung Cancer; Small Intestine Cancer; Soft Tissue Sarcoma, Adult; Soft Tissue Sarcoma, Childhood; Squamous Neck Cancer with Occult Primary, Metastatic; Stomach (Gastric) Cancer; Stomach (Gastric) Cancer, Childhood; Supratentorial Primitive Neuroectodermal Tumors, Childhood; T-Cell Lymphoma, Cutaneous; Testicular Cancer; Thymoma, Childhood; Thymoma, Malignant; Thyroid Cancer; Thyroid Cancer, Childhood; Transitional Cell Cancer of the Renal Pelvis and Ureter; Trophoblastic Tumor, Gestational; Unknown Primary Site, Cancer of, Childhood; Unusual Cancers of Childhood; Ureter and Renal Pelvis, Transitional Cell Cancer; Urethral Cancer; Uterine Sarcoma; Vaginal Cancer; Visual Pathway and Hypothalamic Glioma, Childhood; Vulvar Cancer; Waldenstrom's Macro globulinemia; and Wilms' Tumor.

In some embodiments, the cancer can include or involve prostate cancer, lung cancer, colon cancer, rectum cancer, urinary bladder cancer, melanoma, kidney cancer, renal cancer, oral cavity cancer, pharynx cancer, pancreas cancer, uterine cancer, thyroid cancer, skin cancer, head and neck cancer, cervical cancer, ovarian cancer, and/or hematopoietic cancer.

In one aspect, the multispecific compound is administered prior to, simultaneously with, or following chemotherapy, surgical resection of a tumor, or radiation therapy.

In some embodiments, a multispecific compound or trispecific killer engager compound may be administered, for example, from a single dose to multiple doses per week, although in some embodiments the method can be performed by administering a trispecific killer engager compound at a frequency outside this range. In certain embodiments, a multispecific compound or trispecific killer engager compound may be administered from about once per month to about five times per week.

In some embodiments, the method further includes administering one or more additional therapeutic agents. The one or more additional therapeutic agents may be administered before, after, and/or coincident to the administration of a multispecific compound or trispecific killer engager compound. A multispecific compound or trispecific killer engager compound and the additional therapeutic agents may be co-administered. As used herein, "co-administered" refers to two or more components of a combination administered so that the therapeutic or prophylactic effects of the combination can be greater than the therapeutic or prophylactic effects of either component administered alone. Two components may be co-administered simultaneously or sequentially. Simultaneously co-administered components may be provided in one or more pharmaceutical compositions. Sequential co-administration of two or more components includes cases in which the components are administered so that each component can be present at the treatment site at the same time. Alternatively, sequential co-administration of two components can include cases in which at least one component has been cleared from a treatment site, but at least one cellular effect of administering the component (e.g., cytokine production, activation of a certain cell population, etc.) persists at the treatment site until one or more additional components are administered to the treatment site. Thus, a co-administered combination can, in certain circumstances, include components that never exist in a chemical mixture with one another. In other embodiments, the multispecific compound or trispecific killer engager compound and the additional therapeutic agent may be administered as part of a mixture or cocktail. In some aspects, the administration of the multispecific compound or trispecific killer engager compound may allow for the effectiveness of a lower dosage of other therapeutic modalities when compared to the administration of the other therapeutic agent or agents alone, thereby decreasing the likelihood, severity, and/or extent of the toxicity observed when a higher dose of the other therapeutic agent or agents is administered.

The term “chemotherapeutic agent” as used herein refers to any therapeutic agent used to treat cancer. Examples of chemotherapeutic agents include, but are not limited to, actinomycin, azacitidine, azathioprine, bleomycin, bortezomib, carboplatin, capecitabine, cisplatin, chlorambucil, cyclophosphamide, cytarabine, daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, epothilone, etoposide, fluorouracil, gemcitabine, hydroxyurea, idarubicin, imatinib, irinotecan, mechlorethamine, mercaptopurine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, teniposide, tioguanine, topotecan, valrubicin, vinblastine, vincristine, vindesine, vinorelbine, panitumamab, Erbitux™ (cetuximab), matuzumab, IMC-IIF 8, TheraCIM hR3, denosumab, Avastin™ (bevacizumab), Humira™ (adalimumab), Herceptin™ (trastuzumab), Remicade™ (infliximab), rituximab, Synagis™ (palivizumab), Mylotarg™ (gemtuzumab oxogamicin), Raptiva™ (efalizumab), Tysabri™ (natalizumab), Zenapax™ (dacliximab), NeutroSpec™ (Technetium (99mTc) fanolesomab), tocilizumab, ProstaScint™ (Indium-Ill labeled Capromab Pendetide), Bexxar™ (tositumomab), Zevalin™ (ibritumomab tiuxetan (IDEC-Y2B8) conjugated to yttrium 90), Xolair™ (omalizumab), Mab Thera™ (Rituximab), ReoPro™ (abciximab), MabCampath™ (alemtuzumab), Simulect™ (basiliximab), LeukoScan™ (sulesomab), CEA-Scan™ (arcitumomab), Verluma™ (nofetumomab), Panorex™ (Edrecolomab), alemtuzumab, CDP 870, natalizumab Gilotrif™ (afatinib), Lynparza™ (olaparib), Perjeta™ (pertuzumab), Otdivo™ (nivolumab), Bosulif™ (bosutinib), Cabometyx™ (cabozantinib), Ogivri™ (trastuzumab-dkst), Sutent™ (sunitinib malate), Adcetris™ (brentuximab vedotin), Alecensa™ (alectinib), Calquence™ (acalabrutinib), Yescarta™ (ciloleucel), Verzenio™ (abemaciclib), Keytruda™ (pembrolizumab), Aliqopa™ (copanlisib), Nerlynx™ (neratinib), Imfinzi™ (durvalumab), Darzalex™ (daratumumab), Tecentriq™ (atezolizumab), and Tarceva™ (erlotinib). Examples of immunotherapeutic agent include, but are not limited to, interleukins (I1-2, I1-7, I1-12), cytokines (Interferons, G-CSF, imiquimod), chemokines (CCL3, CC126, CXCL7), immunomodulatory imide drugs (thalidomide and its analogues).

In some aspects, the chemotherapy is selected from the group consisting of altretamine, amsacrine, L-asparaginase, colaspase, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, cyclophosphamide, cytophosphane, cytarabine, dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epirubicin, etoposide, fluorouracil, fludarabine, fotemustine, ganciclovir, gemcitabine, hydroxyurea, idarubicin, ifosfamaide, irinotecan, lomustine, melphalan, mercaptopurine, methotrexate, mitoxantrone, mitomycin C, nimustine, oxaliplatin, paclitaxel, pemetrexed, procarbazine, raltitrexed, temozolomide, teniposide, tioguanine, thiotepa, topotecan, vinblastine, vincristine, vindesine, and vinorelbine.

In some embodiments, of the method can include administering sufficient multispecific compound or trispecific killer engager compound as described herein and administering the at least one additional therapeutic agent demonstrates therapeutic synergy. In some aspects of the methods of the present invention, a measurement of response to treatment observed after administering both a multispecific compound or trispecific killer engager compound as described herein and the additional therapeutic agent is improved over the same measurement of response to treatment observed after administering either the multispecific compound or trispecific killer engager compound or the additional therapeutic agent alone. The term “subject” as used herein refers to any individual or subject to which the subject methods are performed. In many embodiments, the subject is human, although the subject may be any non-human animal. Suitable non-human animals include, but are limited to, a vertebrate such as a rodent (including a mouse, a rat, a hamster, or a guinea pig), a cat, a dog, a rabbit, a farm animal (including a cow, a horse, a goat, a sheep, a pig, a chicken, etc.), or a primate (including a monkey, a chimpanzee, an orangutan, or a gorilla).

In some embodiments of this aspect, the anti-B7H3 multispecific compound can include an immune cell activating domain that includes IL-15 or a functional portion thereof, operably linked to the NK engaging domain. In some embodiments, the multispecific anti-B7H3 compound can have the amino acid sequence as set forth in any one of SEQ ID NOs:20-25.

In another aspect, this disclosure describes a chimeric antigen receptor compound that includes one of the anti-B7H3 proteins described herein. Chimeric antigen receptors (CARs, also known as chimeric immunoreceptors, chimeric T cell receptors, or artificial T cell receptors) are receptor proteins that have been engineered to give T cells the new ability to target a specific protein. The receptors are chimeric because they combine both antigen binding and T cell activating functions into a single receptor.

CAR-T cell therapy uses T cells engineered with CARs for cancer therapy. The premise of CAR-T immunotherapy is to modify T cells to recognize cancer cells to target and destroy the cancer cells more effectively. T cells are harvested from a donor (autologous or allogeneic), genetically altered, then the resulting CAR-T cells are infused into a subject to attack the subject's tumor. CAR-T cells can be either derived from T cells in a subject's own blood (autologous) or derived from the T cells of a donor (allogeneic). Once isolated from a person, these T cells are genetically engineered to express a specific CAR that programs the T cells to target an antigen that is present on the surface of a tumor. For safety, CAR-T cells are engineered to be specific to an antigen expressed on a tumor that is not expressed on healthy cells. After CAR-T cells are infused into a subject, they act as a “living drug” against cancer cells. When the CAR-T cells contact their targeted antigen on a cell, CAR-T cells bind to the antigen, become activated, proliferate, and become cytotoxic. CAR-T cells destroy cells through several mechanisms including extensive stimulated cell proliferation, increasing the degree to which they are toxic to other living cells (cytotoxicity), and by inducing increased secretion of factors that can affect other cells (e.g., cytokines, interleukins, and/or growth factors). In another aspect, this disclosure describes a targeted therapeutic compound that includes a targeting domain and a therapeutic domain linked to the targeting domain. The targeting domain includes any embodiment of the anti-B7H3 proteins described herein. In some embodiments, the targeted therapeutic compound can provide immunotherapy and, therefore, be a targeted immunotherapeutic compound. In some embodiments, the therapeutic domain can include a drug, a therapeutic radioisotope, a toxin, a cytokine, or a chemokine.

As used herein, the term “drug” refers to any chemical substance, which, when administered to a living organism, produces a biological effect. A pharmaceutical drug is a chemical substance used to treat, cure, prevent, or diagnose a disease or to promote well-being. Drugs can be obtained through extraction from medicinal plants, or by organic synthesis. Pharmaceutical drugs may be used for a limited duration, or on a regular basis for chronic disorders.

A “radioisotope” or “radionuclide” is an atom that has excess nuclear energy, making it unstable. This excess energy can be used in one of three ways: emitted from the nucleus as gamma radiation; transferred to one of its electrons to release it as a conversion electron; or used to create and emit a new particle (alpha particle or beta particle) from the nucleus. During those processes, the radionuclide is said to undergo radioactive decay. These emissions are considered ionizing radiation because they are powerful enough to liberate an electron from another atom. The radioactive decay can produce a stable nuclide or will sometimes produce a new unstable radionuclide that may undergo further decay.

As used herein, the term “toxin” refers to a substance harmful to cells. Toxins can be small molecules, peptides, or proteins that are capable of causing disease or cell death on contact with, or absorption by, body tissues. Toxins vary greatly in their toxicity. Toxins are largely secondary metabolites, which are organic compounds that are not directly involved in an organism's growth, development, or reproduction, instead often aiding the organism in matters of defense. In some applications, a toxin may be used therapeutically by targeting the effect of the toxin toward an undesirable cell or cells (e.g., tumor cells).

Cytokines are a broad category of small proteins (~5-20 kDa) involved in cell signaling. Cytokines are peptides and cannot cross the lipid bilayer of cells to enter the cytoplasm, but are nevertheless involved in autocrine, paracrine, and endocrine signaling as immunomodulating agents. Cytokines include chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors, but generally not hormones or growth factors (despite some overlap in the terminology). Cytokines are produced by a broad range of cells, including immune cells such as macrophages, B lymphocytes, T lymphocytes, mast cells, endothelial cells, fibroblasts, and various stromal cells. Cytokines modulate the balance between humoral and cell-based immune responses, and they regulate the maturation, growth, and responsiveness of particular cell populations.

In a further aspect, this disclosure describes a targeted imaging compound that includes a targeting domain and an imaging domain linked to the targeting domain. The targeting domain includes any embodiment of one of the anti-B7H3 proteins described herein. The imaging domain can include any moiety that can produce a detectable signal. Exemplary imaging moieties include, but are not limited to a colorimetric label, a fluorescent label, a radioactive label, a magnetic label, or an enzymatic label.

In yet another aspect, this disclosure describes a capture assay device including any embodiment of one of the anti-B7H3 proteins described herein immobilized to a substrate. For example, an anti-B7H3 protein described herein can be incorporated into cell and/or ligand capture technology such as, for example, an ELISA-based assay. A substrate to immobilize the anti-B7H3 proteins can include, for example, a cell culture plate or dish, a glass slide, or any other support than can be used to perform an assay requiring an immobilized anti-B7H3 protein.

In the preceding description and following claims, the term “and/or” means one or all of the listed elements or a combination of any two or more of the listed elements; the terms “comprises,” “comprising,” and variations thereof are to be construed as open ended — i.e., additional elements or steps are optional and may or may not be present; unless otherwise specified, “a,” “an,” “the,” and “at least one” are used interchangeably and mean one or more than one; and the recitations of numerical ranges by endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

In the preceding description, particular embodiments may be described in isolation for clarity. Unless otherwise expressly specified that the features of a particular embodiment are incompatible with the features of another embodiment, certain embodiments can include a combination of compatible features described herein in connection with one or more embodiments. For any method disclosed herein that includes discrete steps, the steps may be conducted in any feasible order. And, as appropriate, any combination of two or more steps may be conducted simultaneously.

Presented below are examples discussing trispecific compounds including a B7H3 binding protein contemplated for the discussed applications. The following examples are provided to further illustrate the embodiments of the present invention but are not intended to limit the scope of the invention. While they are typical of those that might be used, other procedures, methodologies, or techniques known to those skilled in the art may alternatively be used.

EXAMPLES

EXAMPLE 1

Construction of caml615B7H3 Trispecific Killer Engager compounds

The CDR regions from a camelid (llama) anti-CD 16 (Behar et al., Protein Eng Des Sel. 2008;21(1):1-10. doi: 10.1093/protein/gzm064. PubMed PMID: 18073223) were spliced into a universal, humanized, heavy chain scaffold (Vincke et al., J Biol Chem. 2009;284(5):3273-84. doi: 10.1074/jbc.M806889200. PubMed PMID: 19010777). This new, humanized camelid sequence was used to manufacture sdAb B7H3 trispecific killer engager. A hybrid coding region encoding cam 16, an (SGGGG)₄ linker (SEQ ID NO: 27), wild type rhIL-15, a whitlow linker, and the noted (FIG. IB “CD276-new-2", SEQ ID NO:2) anti-B7H3 sdAb was synthesized using Hi- fi DNA cloning techniques. The Biomedical Genomics Center, University of Minnesota, St.

Paul, MN verified the gene sequence and the in-frame accuracy of the construct. Gene products were amplified, and the vector was transfected in Expi-293 cells. Supernatants were isolated 4-7 days later and enriched using HlS-columns in an Akta Pure platform. Protein purity was determined with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) stained with Simply Blue Safe Stain (Invitrogen, Carlsbad, CA).

Cancer Cell Lines

MA-148 (Geller et al., 2013. Cytotherapy 15(10): 1297-1306) is a human epithelial high grade serous ovarian carcinoma cell line. For in vivo experiments, lines were transfected with a luciferase reporter construct using transfection reagent (LIPOFECTAMINE reagent, Invitrogen, Carlsbad, CA) and selective pressure applied with 10 μg/mL of blasticidin. Ovarian carcinoma cells OVCAR-8 (RRID:CVCL_1629) were obtained from the DTP, DCTD Tumor Repository sponsored by the Biological Testing Branch, Developmental Therapeutics Program, NCI, NIH. Other cell lines were obtained from the American Type Culture Collection including C4-2 (prostate; RRID:CVCL_4782), DU145 (prostate; RRID:CVCL_0105), LNCaP (prostate; RRåD:CVCL_0395), PC-3 (prostate; RRID:CVCL_0035), A549 (lung; RRID:CVCL_0023), and NCI-H460 (lung; RRID:CVCL_0459). All lines were maintained in RPMI 1640 RPMI supplemented with 10-20% fetal bovine serum (FBS) and 2 mmol/L L-glutamine. Lines were incubated at a humidified atmosphere containing 5% CO₂ at a constant 37°C. When adherent cells were more than 90% confluent, they were passaged using trypsin-EDTA for detachment. For the cell counts a standard hemocytometer was used. Only those cells with a viability > 95% were used for the experiments, as determined by trypan blue exclusion.

Cell Products

Peripheral blood mononuclear cells (PBMCs) were obtained from normal volunteers or subjects after consent was received and institutional review board (IRB) approval was granted, in compliance with guidelines by the Committee on the Use of Human Subjects in Research and in accordance with the Declaration of Helsinki. Cells were pelleted, lysed for red blood cells, cryopreserved in 10% DMSO/90% FBS and stored in liquid nitrogen.

Evaluation of Cytotoxicity and NK cell Activation

Antibody-dependent cell-mediated cytotoxicity (ADCC) was measured in a flow cytometry assay by evaluating degranulation via CD 107a (lysosomal-associated membrane protein LAMP-1) and intracellular IFN-γ production. Upon thawing, normal donor and subject derived PBMCs or ascites cells were rested overnight (37°C, 5% CO2) in RPMI 1640 media supplemented with 10% fetal calf serum (RPMI- 10). The next morning, they were and suspended with tumor target cells or media after washing twice with RPMI- 10. Cells were then incubated with trispecific killer engager compounds or controls for 10 minutes at 37°C. Fluorescein isothiocyanate (FITC)-conjugated anti -human CD 107a monoclonal antibody (BD Biosciences, San Jose, CA) was then added and incubated for one hour. After incubation, GOLGISTOP (1:1,500, BD Biosciences, San Jose, CA) and GOLGIPLUG (1:1,000, BD Biosciences, San Jose, CA) were added for three hours (37°C, 5% CO2). After washing with phosphate buffered saline, the cells were stained with PE/Cy 7-conjugated anti-CD56 mAh, APC/Cy 7-conjugated anti-CD 16 mAh, and PE-CF594-conjugated anti-CD3 mAh (BioLegend, Inc., San Diego, CA). Cells were incubated for 15 minutes at 4°C, washed, and fixed with 2% paraformaldehyde. Cells were then permeabilized using an intracellular perm buffer (BioLegend, Inc., San Diego, CA) to evaluate production of IFN-gamma (IFN-γ) through detection via aBV650 conjugated anti-human IFN-γ antibody (BioLegend, Inc., San Diego, CA). Samples were washed and the evaluated in an LSRII flow cytometer LSRII flow cytometer (BD Biosciences, San Jose, CA).

Real-time tumor killing assay

Tumor killing was evaluated in real-time using the INCUCYTE platform (Essen Biosciences, Inc., Ann Arbor, MI). Magnetic-bead-enriched 40,000 CD3-CD56⁺NK effector cells were plated into 96-well ULA flat clear bottom polystyrene tissue-culture treated microplates (Coming, Flintshire, UK) along with GFP stably expressing OVCAR8 spheroids, 20,000 cells allowed to establish spheroids for three days prior to co-culture. Noted treatments were then added at a 30 nM concentration and the plate was placed in an INCUCYTE S3 platform (Essen Biosciences, Inc., Ann Arbor, MI) housed inside a cell incubator at 37°C/5% CO₂. Images from three technical replicates were taken every hour for 120 hours using a 4X objective lens and then analyzed using the INCUCYTE basic software (Essen Biosciences, Inc., Ann Arbor, MI). Graphed readouts represent integrated spheroid GFP fluorescence intensity, normalized to tumor alone at the starting (0 hr) time point.

Statistical Analysis

PRISM software (GraphPad Software, Inc., La Jolla, CA) was used to create all statistical tests. For all in vitro studies One-way ANOVA with repeated measures was used to calculate significance in comparisons to the caml615B7H3 group. Bars represent mean ± SEM. Statistical significance is displayed as *P<0.05, **P< 0.01, ***P<0.001, and ****P<0.0001.

EXAMPLE 2 A second-generation trispecific killer engager compound capable of both antibody- dependent cellular cytotoxicity (ADCC) and NK cell expansion was constructed by modifying a previously reported trispecific killer engager compound platform (Vallera et al., Clin Cancer Res. 2016;22(14):3440-50. doi: 10.1158/1078-0432.CCR-15-2710. PubMed PMID: 26847056; PMC ID: PMC4947440; US Patent Application Publication No. 2018/0282386 Al). In an exemplary construct, a wild-type human IL-15 crosslinker with two modified flanking regions was inserted between two antibody fragments: an N-terminal VHH humanized camelid anti- CD 16 fragment and a C-terminal anti-B7H3 VHH, or single domain antibody, creating an anti- B7H3 trispecific killer engager, a schematic illustration of which is shown in FIGURE 1.

To assess the ability of an exemplary anti-B7H3 protein — an sdAb — to target activity of a trispecific killer engager, an anti-B7H3 trispecific NK engager molecule was used to induce NK cell activity against B7H3 -containing targets: prostate cancer cell lines, lung cancer cells lines, and ovarian cancer cell lines. Generally, trispecific NK engager molecules recruit and activate NK cells against a targeted cell population. NK activation can be measured, for example, using flow cytometric assays that measure natural killer (NK) cell degranulation (detecting CD107a⁺ NK cells) and inflammatory cytokine production (e.g., IFN-γ).

Exemplary anti-B7H3 protein sequences were incorporated into a trispecific killer engager backbone and compared to a trispecific killer engager incorporating an anti-B7H3 scFv sequence. NK cell degranulation (measured as the percent CD107a⁺ NK cells) and interferon gamma (IFN-γ) production (measured as the percent of IFN-y⁺ NK cells) were evaluated by flow cytometry as described in Example 1 in various cancer cell lines, and in the presence of various concentration of trispecific killer engager. Briefly, the trispecific killer engager compounds were incubated for five hours before NK cell degranulation and inflammatory cytokine production (IFN-γ) was measured by flow cytometry. The results presented reflect the average of N=3 independent experiments.

The exemplary anti-B7H3 proteins incorporated into trispecific killer engager molecules enhanced NK cell degranulation and IFN-γ production against prostate cancer cells, as demonstrated in PC3 and DU145 cells (see Figures 2B, 2C, 2E and 2F), and LnCAP and C4-2 cells (see Figures 3A, 3B, 3C and 3D), when co-cultured with peripheral blood mononuclear cells (PBMCs) at a 30nM concentration; as compared to PBMCs incubated alone (see Figures 2 A and 2D). Figure 2 and Figure 3 provide data showing that trispecific NK engager molecules containing an exemplary anti-B7H3 sdAb protein induces activity of the NK cells against a B7H3 -expressing target cell population. The anti-B7H3 trispecific NK engagers induced NK cell degranulation and NK cell inflammatory cytokine production against multiple prostate cancer cells lines. For both measurements of NK activation, the sdAb-containing trispecific NK engager (black bars) induced greater responses than an anti-B7H3 trispecific NK engager molecule containing an anti-B7H3 scFv (gray bars).

The exemplary anti-B7H3 proteins incorporated into trispecific killer engager molecules also enhanced NK cell degranulation and IFN-γ production against lung cancer cells, as demonstrated in A549 and NCI-H460 cells (see Figures 4C, 4F, 5A and 5D), and ovarian cancer cells, as demonstrated in OVCAR8 and MA148 (see Figures 5B, 5C, 5E and 5F) cells, when cocultured with PBMCs at a 30nM concentration; as compared to PBMCs incubated alone (see Figures 4A and 4D) or co-cultured with C4-2 cells (see Figures 4B and 4E).

Figure 4 and Figure 5 provide data showing that trispecific NK engager molecules containing an exemplary anti-B7H3 sdAb protein induces activity of the NK cells against additional B7H3 -expressing target cell populations: prostate cancer cell line C4-2, lung cancer cell lines A549 and NCI-H460, and ovarian cancer cell lines OVCAR8 and MA148. Once again, the sdAb-containing trispecific NK engager (black bars) induced greater responses than an anti- B7H3 trispecific NK engager molecule containing an anti-B7H3 scFv (gray bars).

The exemplary anti-B7H3 proteins incorporated into trispecific killer engager molecules were demonstrated to enhance NK cell degranulation against prostate, at three of the doses tested: 0.3; 3 and 30 nM (see Figures 6D, 6E and 6F) as compared to the absence of effect when incubated with PBMCs alone, independently of the dose (see Figures 6A, 6B and 6C).

Similar effects were observed when IFN-γ production was assessed; the exemplary anti- B7H3 proteins incorporated into trispecific killer engager molecules enhanced IFN-γ production against prostate, at the three doses tested: 0.3; 3 and 30 nM (see Figures 7D, 7E and 7F) as compared to the absence of effect when incubated with PBMCs alone, independently of the dose (see Figures 7A, 7B and 7C).

Figure 6 and Figure 7 provide data showing that trispecific NK engager activity is target dependent. Background NK activity is low in the absence of B7H3 -expressing target cells. NK cell degranulation and IFN-γ production are induced when B7H3 -expressing target cells are co- cultured with PBMC NK cells and anti -B7H3 -containing trispecific engager molecules. Once again, the sdAb-containing trispecific NK engager (black bars) induced greater responses than an anti-B7H3 trispecific NK engager molecule containing an anti-B7H3 scFv (gray bars).

The ability of the trispecific killer engager incorporating exemplary anti-B7H3 protein to enhance cytolytic activity against ovarian cancer spheroids was then assessed in vitro, as described in Example 1. Briefly, an exemplary anti-B7H3 protein was incorporated into a trispecific killer engager backbone and its activity was compared to the activity of NK cells alone in a spheroid assay. 20,000 GFP-expressing OVCAR8 cells were plated in a well of a 96- well ULA plate and allowed to form for three days. 40,000 enriched NK cells were then added, either alone or with 30 nM trispecific killer engager. Pictures showing GFP intensity measurements were taken every hour for 120 hours. It is to be noted that cellular death induces a temporary increase in green fluorescence. Three technical replicates for each of the three biologic replicates were performed. As shown in Figure 8, and as further quantified in Figure 9, the trispecific killer engager incorporating exemplary anti-B7H3 protein enhanced cytolytic activity against ovarian cancer spheroids. As compared to the activity of NK cells alone.

Figure 8 and Figure 9 provide data showing the ability of the anti-B7H3-sdAb-contining trispecific NK engager to kill ovarian cancer cells, using an advanced imaging based cytolytic assay. In this assay, 20,000 OVCAR8 ovarian cancer cells were stably transduced with a GFP cassette (to provide green fluorescence), then plated in an ultra-low adhesion (ULA) 96-well plate to allow spheroids to form over a three-day period. After three days, nothing (Tumor Alone), 40,000 NK cells with a small amount of cytokine to maintain survival (Tumor+NK), or 40,000 NK cells and 30 nM trispecific NK engager containing an anti-B7H3 sdAb are added. Spheroid killing, measured as loss of spheroid green-fluorescence intensity, is observed over a five-day (120 hours) period. When treated with the trispecific NK engager molecule containing the anti-B7H3 sdAb, the NK cells kill the spheroid much better than when no trispecific is present, showing that this trispecific can induce killing of three-dimensional tumor formations.

The complete disclosure of all patents, patent applications, and publications, and electronically available material (including, for instance, nucleotide sequence submissions in, e.g., GenBank and RefSeq, and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq) cited herein are incorporated by reference in their entirety. In the event that any inconsistency exists between the disclosure of the present application and the disclosure(s) of any document incorporated herein by reference, the disclosure of the present application shall govern. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements.

All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.

Although the invention has been described with reference to the above examples, it will be understood that modifications and variations are encompassed within the spirit and scope of the invention. Accordingly, the invention is limited only by the following claims.

Sequence Listing Free Text

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nucleotides 1-9: Kozak sequence nucleotides 10-1296: encode SEQ ID NO:25

Claims

What is claimed is:

1. An anti-B7H3 polypeptide comprising at least one of SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO: 6, or a functional variant thereof.

2. An anti-B7H3 polypeptide comprising SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, a CDR region of SEQ ID NO: 1, a CDR region of SEQ ID NO:2, a CDR region of SEQ ID NO:3, or a functional variant thereof.

3. A multispecific compound comprising: a targeting domain comprising an anti-B7H3 polypeptide, the anti-B7H3 polypeptide comprising:

SEQ ID NO:4,

SEQ ID NO:5,

SEQ ID NO: 6, a CDR region of SEQ ID NO: 1 a CDR region of SEQ ID NO:2, a CDR region of SEQ ID NO: 3, or a functional variant thereof; and an immune cell engaging domain operably linked to the targeting domain.

4. The multispecific compound of claim 3, wherein the immune cell is a T cell or a natural killer (NK) cell.

5. The multispecific compound of claim 4, wherein: the immune cell is an NK cell; and the immune cell engaging domain comprises a ligand or antibody that specifically binds to CD 16.

6. The multispecific compound of claim 5, wherein the antibody that specifically binds to CD 16 comprises an scFv, a F(ab)₂, a Fab, or a single domain antibody (sdAb).

7. The multispecific compound of any one of claims 3-6, wherein: the immune cell engaging domain comprises SEQ ID NO: 19; and the targeting domain comprises SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3.

8. The multispecific compound of claim 7, wherein the targeting domain and the immune cell engager domain are linked by SEQ ID NO: 14.

9. The multispecific compound of claim 7, wherein the compound comprises amino acids 19-294 of SEQ ID NO:20 or amino acids 19-284 of SEQ ID NO:21.

10. The multispecific compound of any one of claims 3-9, further comprising an immune cell activating domain.

11. The multispecific compound of claim 10, wherein: the immune cell comprises an NK cell; and the immune cell activating domain comprises a cytokine or a functional portion thereof.

12. The multispecific compound of claim 11, wherein the cytokine is IL-15 or a functional variant thereof.

13. The multispecific compound of any one of claims 10-12, wherein the compound comprises:

SEQ ID NO: 19;

SEQ ID NO: 11 operably linked to SEQ ID NO: 19; and a targeting domain operably linked to SEQ ID NO: 19 and SEQ ID NO: 11, the targeting domain comprising SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3.

14. The multispecific compound of claim 13, wherein:

SEQ ID NO: 19 and SEQ ID NO: 11 are linked by SEQ ID NO: 14; and

SEQ ID NO: 11 is linked to the targeting domain by and 1 or 2 are linked by SEQ ID

NO:15.

15. The multispecific compound of claim 14, wherein the compound is as set forth as in SEQ ID NO:22 or SEQ ID NO:23.

16. The multispecific compound of claim 12-15, wherein the functional variant of IL-15 comprises an N72D or N72A amino acid substitution compared to SEQ ID NO: 11.

17. An isolated nucleic acid sequence encoding the multispecific compound of any one of claims 3-16.

18. The isolated nucleic acid sequence of claim 17, wherein the sequence is any one of SEQ ID NOs:26-33, or a sequence having 90% identity to any one of SEQ ID NOs:26-33.

19. A protein encoded by any one of the nucleic acid sequences of claim 17.

20. The protein of claim 19, wherein the protein comprises the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, or any amino acid sequence having 90% or greater identity thereto.

21. A host cell comprising the isolated nucleic acid of claim 17 or claim 18.

22. The host cell of claim 21, wherein the cell is a T cell, an NK cell, or a macrophage.

23. A pharmaceutical composition comprising: the multispecific compound of any one of claims 3-16; and a pharmaceutically acceptable carrier.

24. A method comprising: administering to a subject a multispecific compound in an amount effective to induce natural killer (NK)-mediated killing of a cell, the multispecific compound comprising: a targeting domain comprising the anti-B7H3 polypeptide of claim 1 or claim 2; and an NK engaging domain operably linked to the targeting domain.

25. The method of claim 24, wherein the multispecific compound further comprises an immune cell activating domain comprising an IL-15 or a functional portion thereof, operably linked to the NK engaging domain.

26. The method of claim 24, wherein the multispecific compound is as set forth in any one of SEQ ID NOs:20-25.

27. A method for stimulating expansion of natural killer (NK) cells in vivo comprising: administering to a subject an effective amount of multispecific compound comprising: a targeting domain comprising the anti-B7H3 polypeptide of claim 1 or claim 2; and an NK engaging domain operably linked to the anti-B7H3 polypeptide.

28. The method of claim 27, wherein the multispecific compound further comprises an immune cell activating domain comprising an IL-15 or a functional portion thereof, operably linked to the NK engaging domain.

29. The method of claim 27, wherein the multispecific compound is as set forth in any one of SEQ ID NOs:20-25.

30. A method of treating a subject having, or at risk of having cancer comprising: administering to the subject an effective amount of multispecific compound comprising: a targeting domain comprising the anti-B7H3 polypeptide of claim 1 or claim 2; and an NK engaging domain operably linked to the anti-B7H3 polypeptide.

31. The method of claim 30, wherein the multispecific compound further comprises an immune cell activating domain comprising an IL-15 or a functional portion thereof, operably linked to the NK engaging domain.

32. The method of claim 30, wherein the multispecific compound is as set forth in any one of SEQ ID NOs:20-25.

33. The method of claim 30, wherein cancer cells express B7H3.

34. The method of claim 30, wherein the cancer comprises prostate cancer, lung cancer, colon cancer, rectum cancer, urinary bladder cancer, melanoma, kidney cancer, renal cancer, oral cavity cancer, pharynx cancer, pancreas cancer, uterine cancer, thyroid cancer, skin cancer, head and neck cancer, cervical cancer, ovarian cancer, or hematopoietic cancer.

35. The method of claim 30, wherein the multispecific compound is administered prior to, simultaneously with, or following chemotherapy, surgical resection of a tumor, or radiation therapy.

36. The method of claim 35, wherein the chemotherapy comprises altretamine, amsacrine, L- asparaginase, colaspase, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, cyclophosphamide, cytophosphane, cytarabine, dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epirubicin, etoposide, fluorouracil, fludarabine, fotemustine, ganciclovir, gemcitabine, hydroxyurea, idarubicin, ifosfamaide, irinotecan, lomustine, melphalan, mercaptopurine, methotrexate, mitoxantrone, mitomycin C, nimustine, oxaliplatin, paclitaxel, pemetrexed, procarbazine, raltitrexed, temozolomide, teniposide, tioguanine, thiotepa, topotecan, vinblastine, vincristine, vindesine, or vinorelbine.

37. A chimeric antigen receptor compound comprising the anti-B7H3 polypeptide of claim 1 or claim 2.

38. A targeted immunotherapy compound comprising: a targeting domain comprising the anti-B7H3 polypeptide of claim 1 or claim 2; and an immunotherapeutic domain linked to the targeting domain.

39. A targeted therapeutic compound comprising: a targeting domain comprising the anti-B7H3 polypeptide of claim 1 or claim 2; and a therapeutic domain linked to the targeting domain.

40. The targeted therapeutic compound of claim 37, wherein the therapeutic domain comprises a drug, a therapeutic radioisotope, a toxin, a cytokine, or a chemokine.

41. A targeted imaging compound comprising: a targeting domain comprising the anti-B7H3 polypeptide of claim 1 or claim 2; and an imaging domain linked to the targeting domain.

42. The targeted imaging compound of claim 41, wherein the imaging domain comprises a colorimetric label, a fluorescent label, a radioactive label, a magnetic label, or an enzymatic label.

43. A capture assay device comprising the anti-B7H3 polypeptide of claim 1 or claim 2 immobilized to a substrate.